Why are homologous overlaps important for In-Fusion Cloning reactions?
Why are homologous overlaps important for In-Fusion Cloning reactions?
The mechanism for In?Fusion Cloning reactions employs a 3' exonuclease to generate single-stranded 5' overhangs at the termini of linear double-stranded DNA. These DNA fragments are then annealed via complementary 15-nt overlaps at the termini of the insert(s) and a linearized vector. The vector can be linearized by inverse PCR or restriction digest. Restriction digest can be performed with one or more enzymes that generate 5' overhangs (e.g., EcoRI, BamHI), 3' overhangs (e.g., KpnI), or blunt ends (e.g., HpaII).